Developing drugs that target cancer self-renewal pathways will provide new and efficacious treatments for human disease. Importantly, these new drugs must have increased benefit over conventional drug cocktails and be better tolerated by patients with fewer off-target effects. Toward this end, the zebrafish has emerged as both a powerful model of cancer and chemical discovery. Capitalizing on a unique zebrafish model of rhabdomyosarcoma, we have shown previously that zebrafish tumors are molecularly similar to human embryonal rhabdomyosaroma (ERMS), identified that the RAS pathway is active in a majority of human ERMS, and isolated a self-renewing cell type in this disease. Here, we report new transgenic approaches that target fluorescent proteins to specific tumor cell populations and can visualize myf5-GFP+ tumor-initiating cells in vivo and in real-time. Moreover, zebrafish ERMS respond to drugs in a similar manner as human ERMS, and we establish that both the mTOR and MAP-kinase pathway are critical for continued ERMS growth. One of the limitations of our current zebrafish ERMS model is that each diseased animal must be created by microinjection of transgenic DNA into one-cell stage animals. We propose to optimize cell transplantation of zebrafish ERMS into larvae which would create a large reserve of animals affected with ERMS. Additionally, conditional transgenic approaches will be developed that target GAL4-VP16 to developing muscle populations while a second transgene will drive expression of activated RAS by the UAS promoter. By crossing these two transgenic lines, RAS will be specifically expressed in muscle and lead to ERMS. Aim 2 will focus on using chemical genetic approaches to identify FDA approved drugs that modulate ERMS growth and alter the overall numbers of tumor-initiating cells. ERMS affected fish will be treated with FDA approved compounds and assessed for reduced tumor growth compared with control animals. For the subset of drugs that curb tumor growth, ERMS tumor-initiating cells will be directly visualized in live animals to assess drug effects on the ability to kill ERMS tumor-initiating cells. Lastly, chemicals that both curb tumor growth and reduce the number of self-renewing cells within the zebrafish tumor mass will be assessed for efficacy in human ERMS cell lines. In total, our experiments provide novel methods to create large numbers of zebrafish with ERMS and to directly visualize tumor growth and self-renewal in vivo. Our fluorescent transgenic zebrafish model of embryonal rhabdomyosarcoma when coupled with chemical genetic approaches will uncover FDA approved drugs with anti-tumor activity that specifically ablate self-renewing myf5+ ERMS-initiating cells. PUBLIC HEALTH RELEVANCE: Identification of FDA approved drugs that suppress cancer self-renewal will provide new drug combinations for the treatment of human malignancy. Using a zebrafish model of embryonal rhabdomyosarcoma and chemical genetic approaches, we will identify drugs that specifically target self-renewing ERMS cells for destruction.